PROJECT SUMMARY Effective immune responses require a finely tuned balance of positive and negative signals that support T cell activation, effector development, and protective memory generation, and at the same time provide means for timely termination to prevent pathology. Our studies of murine models of chronic virus infection and aggressive melanoma indicate that the adhesion receptor, PSGL-1 (P-selectin glycoprotein ligand-1, Selplg), a ligand for the selectin family of C-type lectins (E, P, and L), unexpectedly acts as a key negative regulator of CD4 and CD8 effector T cells. Our goals are to generate novel tools that will enable us to identify cell intrinsic mechanisms by which PSGL-1 impacts the responses of T cells as well as other hematopoietic cells that participate in immune regulation and to determine if PSGL-1 represents a new immune checkpoint that can be targeted by mAb blocking to improve immunity in the settings of chronic virus infection and cancer. In the murine model of chronic virus infection with Clone 13 LCMV, which has been widely demonstrated to have clinical relevance to human chronic virus infections (e.g. HIV, HCV, and HBV), we discovered that PSGL-1 deficiency unexpectedly supported viral clearance. This striking outcome was attributable to enhanced T cell intrinsic survival in addition to dramatically increased effector functions of both CD4 and CD8 T cells that was associated reduced expression of the immune inhibitory receptors, PD-1, BTLA, and CD160 that in combination contribute an ?exhausted? state. In exhausted CD8 T cells, PSGL-1 inhibits TCR signals, upregulates PD-1, and limits responsiveness to the ?c family cytokines, IL-7 and IL-2. To gain further insights into roles of PSGL-1 in regulating immune responses in which effector T cells become disabled, we analyzed the responses of PSGL-1 KO mice to an aggressive melanoma line derived from a genetic inducible model that recapitulates many aspects of the human cancer. We find high PSGL-1 levels on tumor infiltrating T cells, dendritic cells, macrophages, and myeloid-derived suppressor cells. Tumor growth was delayed with in PSGL- 1 deficient mice, with preservation of cytokine production by CD4 and CD8 T cells that was linked to lower expression of PD-1. We hypothesize that PSGL-1 can act as a ?brake? that cooperates with immune inhibitory mechanisms to limit the numbers and responses of CD4 and CD8 T cells, which in the contexts of chronic virus infection and cancer contributes to the inability to generate a productive effector response. To test this hypothesis, we propose to develop a PSGL-1fl/fl mouse line that can be used to study conditional and inducible genetic deficiency of PSGL-1 in subsets of T cells and innate cells and to develop PSGL-1 blocking mAb to determine if loss of PSGL-1 engagement after chronic infection or melanoma become established can support reversal of T cell exhaustion an inhibit the expression of PDL-1 and other inhibitory receptors. These tools will enable proof-of-concept studies to determine if targeting PSGL-1 can be a clinical approach to augment adaptive immunity and the mechanisms that contribute to its underappreciated roles in immune regulation.